One-third of costs associated with hospital acquired infections (HAIs) are attributed to surgical site infections (SSIs), and SSIs result in a two-fold increase in patient mortality rates. SSIs are most often caused by flora from the patients skin that enter the body at the incision site and replicate rapidly to cause infection. Although current antiseptics are 99.999% effective at killing bacteria on patients skin, the remaining organisms are disproportionately drug-resistant, and may cause life-threatening infections in patients. Traditionally, efforts to improve upon existing antiseptics have involved introduction of new active biocidal agents that provide only incremental improvements over existing products. Agile Sciences co-founder, Dr. Christian Melander, has discovered a unique library of non-microbicidal molecules that effectively inhibit bacterial defense mechanisms so that bacteria are significantly more susceptible to antimicrobials. These compounds, based on a 2-aminoimidazole (2-AI) subunit, target the response regulator (RR) of bacteria's two component systems (TCSs) so that bacteria are unable to respond and adapt to external insults, such as antimicrobials. Agile's 2-AI molecules have been synthetically optimized to enhance the efficacy of microbicidal products, including chlorhexidine gluconate (CHG), which is the most widely-used active ingredient in pre-operative antiseptics. Notably, the 2-AI compounds are effective against both gram-positive and gram-negative bacteria, and preliminary toxicity tests indicate that these compounds are non-toxic to mammalian cells and multicellular organisms. The goal of this work is to assess the ability of Agile's 2-AI compounds to enhance biocidal activity of the CHG active ingredient in antiseptics using in vitro assays designed to mimic pre-operative antisepsis conditions. In Specific Aim #1, the synergistic activity of 2-AI molecules in conjunctio with CHG will be evaluated in a rapid kill assay, and then active compounds will be incorporated into an antiseptic formulation and evaluated on a skin-like surface. Compound formulations that successfully enhance the activity of CHG toward at least 5 of the 6 most prevalent bacteria in SSIs will be further assessed in vitro for toxicity and irritation potential in Specific Aim #2. Th metric of success of this Phase I proposal is to identify at least one 2-AI compound that provides a minimum of 10-fold enhancement in cidal activity of CHG toward at least 5 of the bacteria tested and also displays acceptable toxicity and skin irritation profiles. Compounds that meet this metric will be further optimized in a Phase II study with the goal of providing a more effective pre-operative antiseptic treatment that will substantially reduce the occurrence of SSIs. PUBLIC HEALTH RELEVANCE: Surgical site infections afflict 290,000 hospital patients in the US each year, resulting in increased morbidity and mortality. Agile Sciences is developing an improved antiseptic product that incorporates a new class of potent small molecules to enhance the ability of the antiseptic to eradicate pathogenic bacteria.